As a prelude to long-term studies to characterize the microbiota of the turkey ceca, 14 DNA isolation protocols were evaluated for their ability to reproducibly characterize microbial diversity. Eight commercially available DNA extraction kits were assessed. DNA quantity and quality were assessed and competitive PCR was used to quantify the 16S bacterial rRNA genes. The Invitrogen Easy-DNA Kit extraction method for large samples yielded over eight times more DNA than any other method (3144 +/- 873 mu g g(-1) of sample, P < 0.05). Bacterial and fungal species richness was estimated by Automated Ribosomal Intergenic Spacer Analysis. The Invitrogen Easy-DNA Kit generated the greatest bacterial species richness (46 +/- 7 peaks) while Bio-Rad Aquapure yielded the highest fungal species richness (71 +/- 9.5 peaks). Cluster analysis indicated different DNA extraction methods generated different microbial community compositions using the same cecal matrix from a single donor bird. Optimized DNA extraction protocols Invitrogen Easy-DNA Kit extraction method for large samples and Bio-Rad Aquapure outperform other methods for extraction of DNA from poultry fecal samples, although these methods do not necessarily recover all available DNA. They will be used in future studies to monitor the dynamics of microbial communities of the avian ceca.